Adjuvanted Influenza Vaccines for Pediatric Use

ABSTRACT

An influenza vaccine adjuvanted with a sub-micron oil-in-water emulsion elicits significantly higher immune responses in human pediatric populations. Compared to an existing unadjuvanted pediatric influenza vaccine, the adjuvanted vaccines provided herein can induce in children a longer persistence of high serum antibody titers and also longer seroconversion and seroprotection. The improvement in immune responses is seen for both influenza A virus and influenza B virus strains, but it is particularly marked for influenza B virus. Moreover, while the existing vaccine provides poor immunity in children after a single dose, the adjuvanted vaccine provides high seroprotection rates against the influenza A virus H3N2 subtype even after a single dose. Furthermore, the adjuvanted vaccine offers significantly better seroprotection against mismatched strains of influenza A virus.

This application is a divisional of U.S. patent application Ser. No.12/378,929 filed Feb. 20, 2009, which claims priority from provisionalapplication 61/066,791, filed Feb. 22, 2008, the complete contents ofboth of which are incorporated in full herein by reference.

TECHNICAL FIELD

This invention is in the field of adjuvanted vaccines for protectingagainst influenza virus infection.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 223002117410SeqList.txt,date recorded: Jun. 20, 2013, size: 1 KB).

BACKGROUND ART

Influenza vaccines currently in general use are described in chapters 17& 18 of reference 1. They are based on live virus or inactivated virus,and inactivated vaccines can be based on whole virus, ‘split’ virus oron purified surface antigens (including haemagglutinin andneuraminidase).

The burden of influenza in healthy young children has been increasinglyrecognized along with new studies on the medical [2-7] and thesocioeconomic [8] impact of influenza. Moreover, children have thehighest attack rates of influenza during epidemic periods, and transmitinfluenza viruses in the community to the high risk groups [8,9].

The American Advisory Committee on Immunization Practices (ACIP) in 2006recommended annual influenza vaccination for all children aged 6-59months, because children aged 6-23 months are at substantially increasedrisk for influenza-related hospitalizations [2-7] and children aged24-59 months are at increased risk for influenza-related clinic andemergency department visits [6]. In July 2008 the ACIP further extendedthe recommendation for seasonal influenza vaccination in adolescentsaged 5 to 18 years [10]. In Europe, some countries have issued similarrecommendations, although the European CDC has taken a more restrictedposition with regard to universal immunization of young children, notingthat efficacy in children under 24 months of age has been insufficientlydocumented and might be as low as 37% [11]. A Cochrane analysis statedthat “the field efficacy of influenza vaccine in young children is notdifferent from placebo” [12]

In addition to modest efficacy, conventional vaccines do not appear toinduce satisfactory protective antibodies in unprimed children,especially the very young ones. More specifically, conventional vaccinesgenerally show lower immunogenicity against the influenza B strain thanagainst influenza A strains [13,14]. ACIP has since 2004 recommended atwo-dose vaccination regimen in immunologically naïve very youngchildren, but more recently such recommendation has been extended tochildren aged up to 8 years of age, because of the accumulating evidenceindicating that 2 doses are required for protection in this population[15].

An additional problem in immunizing children against influenza comesfrom ‘antigenic drift’. Influenza viruses routinely undergo intenseselection to evade the host immune system, resulting in geneticvariation and the generation of novel strains (‘antigenic drift’). Ithas been suggested that antigenic drift is associated with a more severeand early onset of influenza epidemic, since the level of pre-existingimmunity to the drifted strain is reduced to the drifted strain [16].While all three virus strains currently included in seasonal influenzavaccines are subject to antigenic drift, the A/H3N2 strain is known todrift more frequently and new variants tend to replace old ones [17,18].

The pace of antigenic drift can exceed the pace of vaccine manufacture.When a vaccine is released, therefore, the vaccine strains may no longerbe a good match for the circulating strains. A vaccine mismatch canresult in a significant excess of influenza-related mortality, sincevaccine effectiveness is reduced [19]. Vaccine mismatch is a potentiallylarger problem in the most influenza susceptible populations,particularly in young children who do not have pre-existing immunityagainst any influenza viruses. This was shown more recently in the2003/2004 season by the emergence of a drifted mismatch strain(A/Fujian, H3N2), which was not included in the vaccine, and resulted in3 times as many children being hospitalized in intensive care inCalifornia, compared with the previous season [20]. In contrast to youngchildren, the elderly at least have a significant history of priorexposure to circulating influenza strains, which offers them some degreeof cross protection. Drifted influenza strains which emerge aftervaccine recommendations are finalized, as occurred in 1997 and 2003, area significant threat to vaccine-naïve young children.

It is an object of the invention to provide influenza vaccines that areeffective in children, that adequate influenza B virus immunogenicity(to induce an adequate immune response), that give useful protectionagainst common circulating influenza viruses even after a single dose,and/or that are effective in children against drifted influenza A virusstrains, particularly A/H3N2 strains.

SUMMARY OF THE INVENTION

It has now been found that an influenza vaccine adjuvanted with asub-micron oil-in-water emulsion elicits significantly improved immuneresponses in human pediatric populations. Compared to an existingunadjuvanted pediatric influenza vaccine the adjuvanted vaccinesprovided herein can induce in children a longer persistence of highserum antibody titers and also longer seroconversion and seroprotection.The improvement in immune responses is seen for both influenza A virusand influenza B virus strains, but it is particularly marked forinfluenza B virus. Moreover, while the existing vaccine provides poorimmunity in children after a single dose, the adjuvanted vaccineprovides high seroprotection rates against the influenza A virus H3N2subtype even after a single dose. Furthermore, the adjuvanted vaccineoffers significantly better seroprotection against mismatched strains ofinfluenza A virus.

Thus the invention provides an influenza vaccine for use in a child,comprising: (i) an influenza virus antigen; and (ii) an adjuvant.

The invention also provides an immunogenic composition for use inimmunizing a child, wherein the composition comprises: (i) an influenzavirus antigen; and (ii) an adjuvant.

The invention also provides an immunogenic composition for immunizing achild, wherein the composition comprises: (i) an influenza virusantigen; and (ii) an adjuvant.

The invention also provides (i) an influenza virus antigen and (ii) anadjuvant, in the manufacture of an immunogenic composition forimmunizing a child.

The invention also provides a method for raising an immune response in achild, comprising a step of administering to the child an immunogeniccomposition comprising: (i) an influenza virus antigen; and (ii) anadjuvant. Preferably this step is performed on a particular child onlyonce per influenza season.

The invention also provides a composition in unit dosage form, wherein:the composition comprises (i) an influenza virus antigen and (ii) anadjuvant; and the unit dosage has a volume less than 0.5 ml e.g. avolume of between 0.2 ml and 0.3 ml, for example about 0.25 ml.

The invention also provides a composition in unit dosage form, wherein:the composition comprises (i) an influenza virus antigen and (ii) anadjuvant; and the unit dosage contains between 6 and 9 μg of influenzahemagglutinin per influenza virus strain e.g. between 7-8 μg strain, orabout 7.5 μg/strain.

The child being immunized may be aged between 0 months and 36 monthse.g. between 6 months and 35 months, between 6 months and 30 months,between 6 months and 24 months, between 6 months and 23 months (allinclusive). Immunization is ideal after a child is 6 months old butbefore their third birthday, as described in more detail below. Theinvention can also be used with older children e.g. up to 72 months ofage. Thus the child may be between 6 and 72 months old, etc. and so avaccine may be administered before a child's sixth birthday.

The invention is particularly useful for raising a useful immuneresponse against subtype H3N2 of influenza A virus after a single dose,and against both subtype H1N1 of influenza A virus and influenza B virusafter two doses. It may also be used to provide immunity againstpandemic strains. The invention is particularly useful in protectingagainst drifted strains of influenza A virus.

An adjuvanted vaccine that can be used according to the invention is theFLUAD™ product, which is already available but is approved for use onlyin elderly subjects i.e. subjects at least 65 years of age (or, in someregions, at least 60 years of age). The adjuvant in this vaccine is asub-micron oil-in-water emulsion known as MF59. The adjuvant in FLUAD™helps to overcome the age-related immuno-senescence seen in the elderly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows GMRs. In each of the six groups of 3 bars the values arethe ratios of GMTs at (i) day 29, (ii) day 50 and (iii) day 209, againstthe GMT at day 1. The six groups are in three pairs, each pair beingwith (+) or without (−) adjuvant. The three pairs are from left toright: H1N1, H3N2 and B. The horizontal bar shows the CPMP criterion foradult vaccines. Two of the + bars exceed the vertical axis: with H1N1the GMR at day 50 was 33; with H3N2 it was 61.

FIG. 2 shows SC or SI rates, arranged as in FIG. 1, but each group ofthree bars shows percentages at days 29, 50 and 209.

FIG. 3 shows seroprotection rates, arranged as in FIG. 1, but each groupof four bars showing percentages at days 1, 29, 50 and 209.

FIG. 4 shows seroprotection rates (% of subjects) in patients at (i) day50 and (ii) day 209. For each pair of figures, the left-hand bar is theadjuvanted group and the right-hand bar is unadjuvanted. The stars (*)denote P<0.001 versus the unadjuvanted group.

DETAILED DESCRIPTION The Influenza Virus Antigen

The invention uses an influenza virus antigen to immunize a child. Theantigen will typically be prepared from influenza virions but, as analternative, antigens such as haemagglutinin can be expressed in arecombinant host (e.g. in an insect cell line using a baculovirusvector) and used in purified form [21,22]. In general, however, antigenswill be from virions.

The antigen may take the form of a live virus or, more preferably, aninactivated virus. Chemical means for inactivating a virus includetreatment with an effective amount of one or more of the followingagents: detergents, formaldehyde, formalin, β-propiolactone, or UVlight. Additional chemical means for inactivation include treatment withmethylene blue, psoralen, carboxyfullerene (C60) or a combination of anythereof. Other methods of viral inactivation are known in the art, suchas for example binary ethylamine, acetyl ethyleneimine, or gammairradiation. The INFLEXAL™ product is a whole virion inactivatedvaccine.

Where an inactivated virus is used, the vaccine may comprise wholevirion, split virion, or purified surface antigens (includinghemagglutinin and, usually, also including neuraminidase).

An inactivated but non-whole cell vaccine (e.g. a split virus vaccine ora purified surface antigen vaccine) may include matrix protein, in orderto benefit from the additional T cell epitopes that are located withinthis antigen. Thus a non-whole cell vaccine (particularly a splitvaccine) that includes haemagglutinin and neuraminidase may additionallyinclude M1 and/or M2 matrix protein. Useful matrix fragments aredisclosed in reference 23. Nucleoprotein may also be present.

Virions can be harvested from virus-containing fluids by variousmethods. For example, a purification process may involve zonalcentrifugation using a linear sucrose gradient solution that includesdetergent to disrupt the virions. Antigens may then be purified, afteroptional dilution, by diafiltration.

Split virions are obtained by treating purified virions with detergentsand/or solvents to produce subvirion preparations, including the‘Tween-ether’ splitting process. Methods of splitting influenza virusesare well known in the art e.g. see refs. 24-29, etc. Splitting of thevirus is typically carried-out by disrupting or fragmenting whole virus,whether infectious or non-infectious with a disrupting concentration ofa splitting agent. The disruption results in a full or partialsolubilisation of the virus proteins, altering the integrity of thevirus. Preferred splitting agents are non-ionic and ionic (e.g.cationic) surfactants. Suitable splitting agents include, but are notlimited to: ethyl ether, polysorbate 80, deoxycholate, tri-N-butylphosphate, alkylglycosides, alkylthioglycosides, acyl sugars,sulphobetaines, betaines, polyoxyethylenealkylethers,N,N-dialkyl-Glucamides, Hecameg, alkylphenoxy-polyethoxyethanols,quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammoniumbromides), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammoniumsalts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxypolyoxyethanols (e.g. the Triton surfactants, such as Triton X-100 orTriton N101), nonoxynol 9 (NP9) Sympatens-NP/090,) polyoxyethylenesorbitan esters (the Tween surfactants), polyoxyethylene ethers,polyoxyethlene esters, etc. One useful splitting procedure uses theconsecutive effects of sodium deoxycholate and formaldehyde, andsplitting can take place during initial virion purification (e.g. in asucrose density gradient solution). Thus a splitting process can involveclarification of the virion-containing material (to remove non-virionmaterial), concentration of the harvested virions (e.g. using anadsorption method, such as CaHPO₄ adsorption), separation of wholevirions from non-virion material, splitting of virions using a splittingagent in a density gradient centrifugation step (e.g. using a sucrosegradient that contains a splitting agent such as sodium deoxycholate),and then filtration (e.g. ultrafiltration) to remove undesiredmaterials. Split virions can usefully be resuspended in sodiumphosphate-buffered isotonic sodium chloride solution. The BEGRIVAC™,FLUARIX™, FLUZONE™ and FLUSHIELD™ products are split vaccines.

Purified surface antigen vaccines comprise the influenza surfaceantigens haemagglutinin and, typically, also neuraminidase. Processesfor preparing these proteins in purified form are well known in the art.The FLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are subunit vaccines.

Another form of inactivated influenza antigen is the virosome [30](nucleic acid free viral-like liposomal particles). Virosomes can beprepared by solubilization of influenza virus with a detergent followedby removal of the nucleocapsid and reconstitution of the membranecontaining the viral glycoproteins. An alternative method for preparingvirosomes involves adding viral membrane glycoproteins to excess amountsof phospholipids, to give liposomes with viral proteins in theirmembrane. The invention can be used to store bulk virosomes. as in theINFLEXAL V™ and INVAVAC™ products. In some embodiments, the influenzaantigen is not in the form of a virosome.

The influenza virus may be attenuated. The influenza virus may betemperature-sensitive. The influenza virus may be cold-adapted. Thesethree features are particularly useful when using live virus as avaccine antigen.

HA is the main immunogen in current inactivated influenza vaccines, andvaccine doses are standardised by reference to HA levels, typicallymeasured by SRID. Existing vaccines typically contain about 15 μg of HAper strain, although lower doses can be used e.g. for children, or inpandemic situations, or when using an adjuvant. Fractional doses such as½ (i.e. 7.5 μg HA per strain), ¼ and ⅛ have been used, as have higherdoses (e.g. 3× or 9× doses [31,32]). Thus vaccines may include between0.1 and 150 μg of HA per influenza strain, preferably between 0.1 and 50μg e.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc.Particular doses include e.g. about 45, about 30, about 15, about 10,about 7.5, about 5, about 3.8, about 1.9, about 1.5, etc. per strain. Adose of 7.5 μg per strain is ideal for use in children.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content, and a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10⁶⁸-10⁷⁵) per strain is typical.

Influenza virus strains for use in vaccines change from season toseason. In the current inter-pandemic period, vaccines typically includetwo influenza A strains (H1N1 and H3N2) and one influenza B strain, andtrivalent vaccines are typical. The invention may also use viruses frompandemic strains (i.e. strains to which the vaccine recipient and thegeneral human population are immunologically naïve), such as H2, H5, H7or H9 subtype strains (in particular of influenza A virus), andinfluenza vaccines for pandemic strains may be monovalent or may bebased on a normal trivalent vaccine supplemented by a pandemic strain.Depending on the season and on the nature of the antigen included in thevaccine, however, the invention may protect against one or more ofinfluenza A virus hemagglutinin subtypes H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, H15 or H16. The virus may additionally haveany of NA subtypes N1, N2, N3, N4, N5, N6, N7, N8 or N9.

The invention can be used with pandemic influenza A virus strains.Characteristics of a pandemic strain are: (a) it contains a newhemagglutinin compared to the hemagglutinins in currently-circulatinghuman strains, i.e. one that has not been evident in the humanpopulation for over a decade (e.g. H2), or has not previously been seenat all in the human population (e.g. H5, H6 or H9, that have generallybeen found only in bird populations), such that the vaccine recipientand the general human population are immunologically naïve to thestrain's hemagglutinin; (b) it is capable of being transmittedhorizontally in the human population; and (c) it is pathogenic tohumans. Pandemic strains H2, H5, H7 or H9 subtype strains e.g. H5N1,H5N3, H9N2, H2N2, H7N1 and H7N7 strains. Within the H5 subtype, a virusmay fall into a number of clades e.g. clade 1 or clade 2. Six sub-cladesof clade 2 have been identified with sub-clades 1,2 and 3 having adistinct geographic distribution and are particularly relevant due totheir implication in human infections.

Influenza B virus currently does not display different HA subtypes, butinfluenza B virus strains do fall into two distinct lineages. Theselineages emerged in the late 1980s and have HAs which can beantigenically and/or genetically distinguished from each other [33].Current influenza B virus strains are either B/Victoria/2/87-like orB/Yamagatall6/88-like. These strains are usually distinguishedantigenically, but differences in amino acid sequences have also beendescribed for distinguishing the two lineages e.g. B/Yamagatall6/88-likestrains often (but not always) have HA proteins with deletions at aminoacid residue 164, numbered relative to the ‘Lee40’ HA sequence [34]. Theinvention can be used with antigens from a B virus of either lineage (orboth).

Compositions may include antigen(s) from one or more (e.g. 1, 2, 3, 4 ormore) influenza virus strains, including influenza A virus and/orinfluenza B virus. Trivalent vaccines are most typical for use with theinvention, as described above, but in some embodiments a compositionincludes antigen from two influenza A virus strains and two influenza Bvirus strains (e.g. a tetravalent “ABBA” vaccine), for example withhemagglutinin from: (i) a A/H1N1 strain; (ii) a A/H3N2 strain; (iii) aB/Victoria/2/87-like strain; and (iv) B/Yamagata/16/88-like strain.Where a vaccine includes more than one strain of influenza, thedifferent strains are typically grown separately and are mixed after theviruses have been harvested and antigens have been prepared. Thus aprocess of the invention may include the step of mixing antigens frommore than one influenza strain.

An influenza virus used with the invention may be a reassortant strain,and may have been obtained by reverse genetics techniques. Reversegenetics techniques [e.g. 35-39] allow influenza viruses with desiredgenome segments to be prepared in vitro using plasmids. Typically, itinvolves expressing (a) DNA molecules that encode desired viral RNAmolecules e.g. from poll promoters or bacteriophage RNA polymerasepromoters, and (b) DNA molecules that encode viral proteins e.g. frompal promoters, such that expression of both types of DNA in a cell leadsto assembly of a complete intact infectious virion. The DNA preferablyprovides all of the viral RNA and proteins, but it is also possible touse a helper virus to provide some of the RNA and proteins.Plasmid-based methods using separate plasmids for producing each viralRNA can be used [40-42], and these methods will also involve the use ofplasmids to express all or some (e.g. just the PB1, PB2, PA and NPproteins) of the viral proteins, with up to 12 plasmids being used insome methods. To reduce the number of plasmids needed, a recent approach[43] combines a plurality of RNA polymerase 1 transcription cassettes(for viral RNA synthesis) on the same plasmid (e.g. sequences encoding1, 2, 3, 4, 5, 6, 7 or all 8 influenza A vRNA segments), and a pluralityof protein-coding regions with RNA polymerase II promoters on anotherplasmid (e.g. sequences encoding 1, 2, 3, 4, 5, 6, 7 or all 8 influenzaA mRNA transcripts). Preferred aspects of the reference 43 methodinvolve: (a) PB1, PB2 and PA mRNA-encoding regions on a single plasmid;and (b) all 8 vRNA-encoding segments on a single plasmid. Including theNA and HA segments on one plasmid and the six other segments on anotherplasmid can also facilitate matters.

As an alternative to using poll promoters to encode the viral RNAsegments, it is possible to use bacteriophage polymerase promoters [44].For instance, promoters for the SP6, T3 or T7 polymerases canconveniently be used. Because of the species-specificity of pollpromoters, bacteriophage polymerase promoters can be more convenient formany cell types (e.g. MDCK), although a cell must also be transfectedwith a plasmid encoding the exogenous polymerase enzyme.

In other techniques it is possible to use dual poll and polll promotersto simultaneously code for the viral RNAs and for expressible mRNAs froma single template [45,46].

Thus an influenza A virus may include one or more RNA segments from aA/PR/8/34 virus (typically 6 segments from A/PR/8/34, with the HA and Nsegments being from a vaccine strain, i.e. a 6:2 reassortant). It mayalso include one or more RNA segments from a A/WSN/33 virus, or from anyother virus strain useful for generating reassortant viruses for vaccinepreparation. An influenza A virus may include fewer than 6 (i.e. 0, 1,2, 3, 4 or 5) viral segments from an AA/6/60 influenza virus (A/AnnArbor/6/60). An influenza B virus may include fewer than 6 (i.e. 0, 1,2, 3, 4 or 5) viral segments from an AA/1/66 influenza virus (B/AnnArbor/1/66). Typically, the invention protects against a strain that iscapable of human-to-human transmission, and so the strain's genome willusually include at least one RNA segment that originated in a mammalian(e.g. in a human) influenza virus. It may include NS segment thatoriginated in an avian influenza virus.

Strains whose antigens can be included in the compositions may beresistant to antiviral therapy (e.g. resistant to oseltamivir [47]and/or zanamivir), including resistant pandemic strains [48].

HA used with the invention may be a natural HA as found in a virus, ormay have been modified. For instance, it is known to modify HA to removedeterminants (e.g. hyper-basic regions around the cleavage site betweenHA1 and HA2) that cause a virus to be highly pathogenic in avianspecies, as these determinants can otherwise prevent a virus from beinggrown in eggs.

The viruses used as the source of the antigens can be grown either oneggs (e.g. specific pathogen free eggs) or on cell culture. The currentstandard method for influenza virus growth uses embryonated hen eggs,with virus being purified from the egg contents (allantoic fluid). Morerecently, however, viruses have been grown in animal cell culture and,for reasons of speed and patient allergies, this growth method ispreferred.

The cell line will typically be of mammalian origin. Suitable mammaliancells of origin include, but are not limited to, hamster, cattle,primate (including humans and monkeys) and dog cells, although the useof primate cells is not preferred. Various cell types may be used, suchas kidney cells, fibroblasts, retinal cells, lung cells, etc. Examplesof suitable hamster cells are the cell lines having the names BHK21 orHKCC. Suitable monkey cells are e.g. African green monkey cells, such askidney cells as in the Vero cell line [49-51]. Suitable dog cells aree.g. kidney cells, as in the CLDK and MDCK cell lines.

Thus suitable cell lines include, but are not limited to: MDCK; CHO;CLDK; HKCC; 293T; BHK; Vero; MRC-5; PER.C6 [52]; FRhL2; W1-38; etc.Suitable cell lines are widely available e.g. from the American TypeCell Culture (ATCC) collection [53], from the Coriell Cell Repositories[54], or from the European Collection of Cell Cultures (ECACC). Forexample, the ATCC supplies various different Vero cells under catalognumbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and it supplies MDCKcells under catalog number CCL-34. PER.C6 is available from the ECACCunder deposit number 96022940.

The most preferred cell lines are those with mammalian-typeglycosylation. As a less-preferred alternative to mammalian cell lines,virus can be grown on avian cell lines [e.g. refs. 55-57], includingcell lines derived from ducks (e.g. duck retina) or hens. Examples ofavian cell lines include avian embryonic stem cells [55,58] and duckretina cells [56]. Suitable avian embryonic stem cells, include the EBxcell line derived from chicken embryonic stem cells, EB45, EB14, andEB14-074 [59]. Chicken embryo fibroblasts (CEF) may also be used. Ratherthan using avian cells, however, the use of mammalian cells means thatvaccines can be free from avian DNA and egg proteins (such as ovalbuminand ovomucoid), thereby reducing allergenicity.

The most preferred cell lines for growing influenza viruses are MDCKcell lines [60-63], derived from Madin Darby canine kidney. The originalMDCK cell line is available from the ATCC as CCL-34, but derivatives ofthis cell line may also be used. For instance, reference 60 discloses aMDCK cell line that was adapted for growth in suspension culture (‘MDCK33016’, deposited as DSM ACC 2219). Similarly, reference 64 discloses aMDCK-derived cell line that grows in suspension in serum-free culture(‘B-702’, deposited as FERM BP-7449). Reference 65 disclosesnon-tumorigenic MDCK cells, including ‘MDCK-S’ (ATCC PTA-6500),‘MDCK-SF101’ (ATCC PTA-6501), ‘MDCK-SF102’ (ATCC PTA-6502) and ‘MDCK-SF103’ (PTA-6503). Reference 66 discloses MDCK cell lines with highsusceptibility to infection, including ‘MDCK.5F1’ cells (ATCCCRL-12042). Any of these MDCK cell lines can be used.

Virus may be grown on cells in adherent culture or in suspension.Microcarrier cultures can also be used. In some embodiments, the cellsmay thus be adapted for growth in suspension.

Cell lines are preferably grown in serum-free culture media and/orprotein free media. A medium is referred to as a serum-free medium inthe context of the present invention in which there are no additivesfrom serum of human or animal origin. The cells growing in such culturesnaturally contain proteins themselves, but a protein-free medium isunderstood to mean one in which multiplication of the cells occurs withexclusion of proteins, growth factors, other protein additives andnon-serum proteins, but can optionally include proteins such as trypsinor other proteases that may be necessary for viral growth.

Cell lines supporting influenza virus replication are preferably grownbelow 37° C. [67] (e.g. 30-36° C., or at about 30° C., 31° C., 32° C.,33° C., 34° C., 35° C., 36° C.) during viral replication.

Methods for propagating influenza virus in cultured cells generallyincludes the steps of inoculating a culture of cells with an inoculum ofthe strain to be grown, cultivating the infected cells for a desiredtime period for virus propagation, such as for example as determined byvirus titer or antigen expression (e.g. between 24 and 168 hours afterinoculation) and collecting the propagated virus. The cultured cells areinoculated with a virus (measured by PFU or TCID₅₀) to cell ratio of1:500 to 1:1, preferably 1:100 to 1:5, more preferably 1:50 to 1:10. Thevirus is added to a suspension of the cells or is applied to a monolayerof the cells, and the virus is absorbed on the cells for at least 60minutes but usually less than 300 minutes, preferably between 90 and 240minutes at 25° C. to 40° C., preferably 28° C. to 37° C. The infectedcell culture (e.g. monolayers) may be removed either by freeze-thawingor by enzymatic action to increase the viral content of the harvestedculture supernatants. The harvested fluids are then either inactivatedor stored frozen. Cultured cells may be infected at a multiplicity ofinfection (“m.o.i.”) of about 0.0001 to 10, preferably 0.002 to 5, morepreferably to 0.001 to 2. Still more preferably, the cells are infectedat a m.o.i of about 0.01. Infected cells may be harvested 30 to 60 hourspost infection. Preferably, the cells are harvested 34 to 48 hours postinfection. Still more preferably, the cells are harvested 38 to 40 hourspost infection. Proteases (typically trypsin) are generally added duringcell culture to allow viral release, and the proteases can be added atany suitable stage during the culture e.g. before inoculation, at thesame time as inoculation, or after inoculation [67].

In preferred embodiments, particularly with MDCK cells, a cell line isnot passaged from the master working cell bank beyond 40population-doubling levels.

The viral inoculum and the viral culture are preferably free from (i.e.will have been tested for and given a negative result for contaminationby) herpes simplex virus, respiratory syncytial virus, parainfluenzavirus 3, SARS coronavirus, adenovirus, rhinovirus, reoviruses,polyomaviruses, birnaviruses, circoviruses, and/or parvoviruses [68].Absence of herpes simplex viruses is particularly preferred.

Where virus has been grown on a cell line then it is standard practiceto minimize the amount of residual cell line DNA in the final vaccine,in order to minimize any oncogenic activity of the DNA.

Thus a vaccine composition prepared according to the inventionpreferably contains less than 10 ng (preferably less than 1 ng, and morepreferably less than 100 pg) of residual host cell DNA per dose,although trace amounts of host cell DNA may be present.

Vaccines containing <10 ng (e.g. <1 ng, <100 μg) host cell DNA per 15 μgof haemagglutinin are preferred, as are vaccines containing <10 ng (e.g.<1 ng, <100 pg) host cell DNA per 0.25 ml volume. Vaccines containing<10 ng (e.g. <1 ng, <100 pg) host cell DNA per 50 mg of haemagglutininare more preferred, as are vaccines containing <10 ng (e.g. <1 ng, <100pg) host cell DNA per 0.5 ml volume.

It is preferred that the average length of any residual host cell DNA isless than 500 bp e.g. less than 400 bp, less than 300 bp, less than 200bp, less than 100 bp, etc.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination is disclosed in references 69 & 70, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Removal by β-propiolactonetreatment can also be used.

Measurement of residual host cell DNA is now a routine regulatoryrequirement for biologicals and is within the normal capabilities of theskilled person. The assay used to measure DNA will typically be avalidated assay [71,72]. The performance characteristics of a validatedassay can be described in mathematical and quantifiable terms, and itspossible sources of error will have been identified. The assay willgenerally have been tested for characteristics such as accuracy,precision, specificity. Once an assay has been calibrated (e.g. againstknown standard quantities of host cell DNA) and tested then quantitativeDNA measurements can be routinely performed. Three main techniques forDNA quantification can be used: hybridization methods, such as Southernblots or slot blots [73]; immunoassay methods, such as the Threshold™System [74]; and quantitative PCR [75]. These methods are all familiarto the skilled person, although the precise characteristics of eachmethod may depend on the host cell in question e.g. the choice of probesfor hybridization, the choice of primers and/or probes foramplification, etc. The Threshold™ system from Molecular Devices is aquantitative assay for picogram levels of total DNA, and has been usedfor monitoring levels of contaminating DNA in biopharmaceuticals [74]. Atypical assay involves non-sequence-specific formation of a reactioncomplex between a biotinylated ssDNA binding protein, aurease-conjugated anti-ssDNA antibody, and DNA. All assay components areincluded in the complete Total DNA Assay Kit available from themanufacturer. Various commercial manufacturers offer quantitative PCRassays for detecting residual host cell DNA e.g. AppTec™ LaboratoryServices, BioReliance™ Althea Technologies, etc. A comparison of achemiluminescent hybridisation assay and the total DNA Threshold™ systemfor measuring host cell DNA contamination of a human viral vaccine canbe found in reference 76.

The Adjuvant

Compositions of the invention include an adjuvant, which can function toenhance the immune responses (humoral and/or cellular) elicited in apatient who receives the composition. Vaccine adjuvants that can be usedwith the invention include, but are not limited to:

-   -   A mineral-containing composition, including calcium salts and        aluminum salts (or mixtures thereof). Calcium salts include        calcium phosphate (e.g. the “CAP” particles disclosed in ref.        77). Aluminum salts include hydroxides, phosphates, sulfates,        etc., with the salts taking any suitable form (e.g. gel,        crystalline, amorphous, etc.). Adsorption to these salts is        preferred. The mineral containing compositions may also be        formulated as a particle of metal salt [78]. The adjuvants known        as aluminum hydroxide and aluminum phosphate may be used. These        names are conventional, but are used for convenience only, as        neither is a precise description of the actual chemical compound        which is present (e.g. see chapter 9 of reference 162). The        invention can use any of the “hydroxide” or “phosphate”        adjuvants that are in general use as adjuvants. The adjuvants        known as “aluminium hydroxide” are typically aluminium        oxyhydroxide salts, which are usually at least partially        crystalline. The adjuvants known as “aluminium phosphate” are        typically aluminium hydroxyphosphates, often also containing a        small amount of sulfate (i.e. aluminium hydroxyphosphate        sulfate). They may be obtained by precipitation, and the        reaction conditions and concentrations during precipitation        influence the degree of substitution of phosphate for hydroxyl        in the salt. The invention can use a mixture of both an        aluminium hydroxide and an aluminium phosphate. In this case        there may be more aluminium phosphate than hydroxide e.g. a        weight ratio of at least 2:1 e.g. ≧5:1, ≧6:1, ≧7:1, ≧8:1, ≧9:1,        etc. The concentration of Al⁺⁺⁺ in a composition for        administration to a patient is preferably less than 10 mg/ml        e.g. ≦5 mg/ml, ≦4 mg/ml, ≦3 mg/ml, ≦2 mg/ml, ≦1 mg/ml, etc. A        preferred range is between 0.3 and 1 mg/ml. A maximum of 0.85        mg/dose is preferred.    -   Saponins [chapter 22 of ref. 162], which are a heterologous        group of sterol glycosides and triterpenoid glycosides that are        found in the bark, leaves, stems, roots and even flowers of a        wide range of plant species. Saponin from the bark of the        Quillaia saponaria Molina tree have been widely studied as        adjuvants. Saponin can also be commercially obtained from Smilax        ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and        Saponaria officianalis (soap root). Saponin adjuvant        formulations include purified formulations, such as QS21, as        well as lipid formulations, such as ISCOMs. QS21 is marketed as        Stimulon™. Saponin compositions have been purified using HPLC        and RP-HPLC. Specific purified fractions using these techniques        have been identified, including QS7, QS17, QS18, QS21, QH-A,        QH-B and QH-C. Preferably, the saponin is QS21. A method of        production of QS21 is disclosed in ref. 79. Saponin formulations        may also comprise a sterol, such as cholesterol [80].        Combinations of saponins and cholesterols can be used to form        unique particles called immunostimulating complexs (ISCOMs)        [chapter 23 of ref. 162]. ISCOMs typically also include a        phospholipid such as phosphatidylethanolamine or        phosphatidylcholine. Any known saponin can be used in ISCOMs.        Preferably, the ISCOM includes one or more of QuilA, QHA & QHC.        ISCOMs are further described in refs. 80-82. Optionally, the        ISCOMS may be devoid of additional detergent [83]. A review of        the development of saponin based adjuvants can be found in refs.        84 & 85.    -   Bacterial ADP-ribosylating toxins (e.g. the E. coli heat labile        enterotoxin “LT”, cholera toxin “CT”, or pertussis toxin “PT”),        and in particular detoxified derivatives thereof, such as the        mutant toxins known as LT-K63 and LT-R72 [86] or CT-E29H [87].        The use of detoxified ADP-ribosylating toxins as mucosal        adjuvants is described in ref. 88 and as parenteral adjuvants in        ref. 89.    -   Bioadhesives and mucoadhesives, such as esterified hyaluronic        acid microspheres [90] or chitosan and its derivatives [91].    -   Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in        diameter, more preferably ˜200 nm to ˜30 μm in diameter, or ˜500        nm to ˜10 μm in diameter) formed from materials that are        biodegradable and non-toxic (e.g. a poly(α-hydroxy acid), a        polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a        polycaprolactone, etc.), with poly(lactide-co-glycolide) being        preferred, optionally treated to have a negatively-charged        surface (e.g. with SDS) or a positively-charged surface (e.g.        with a cationic detergent, such as CTAB).    -   Liposomes (Chapters 13 & 14 of ref. 162). Examples of liposome        formulations suitable for use as adjuvants are described in        refs. 92-94.    -   Muramyl peptides, such as        N-acetylmuramyl-L-threonyl-D-isoglutamine (“thr-MDP”),        N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),        N-acetylglucsaminyl-N-acetylmuramyl-L-AI-D-isoglu-L-Ala-dipalmitoxy        propylamide (“DTP-DPP”, or “Theramide™),        N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-nipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine        (“MTP-PE”).    -   A polyoxidonium polymer [95,96] or other N-oxidized        polyethylene-piperazine derivative.    -   Methyl inosine 5′-monophosphate (“MIMP”) [97].    -   A polyhydroxlated pyrrolizidine compound [98], such as one        having formula:

-   -   where R is selected from the group comprising hydrogen, straight        or branched, unsubstituted or substituted, saturated or        unsaturated acyl, alkyl (e.g. cycloalkyl), alkenyl, alkynyl and        aryl groups, or a pharmaceutically acceptable salt or derivative        thereof. Examples include, but are not limited to: casuarine,        casuarine-6-α-D-glucopyranose, 3-epi-casuarine, 7-epi-casuarine,        3,7-diepi-casuarine, etc.    -   A CD1d ligand, such as an α-glycosylceramide [99-106] (e.g.        α-galactosylceramide), phytosphingosine-containing        α-glycosylceramides, OCH, KRN7000        [(2S,3S,4R)-1-O-(α-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol],        CRONY-101, 3″-O-sulfo-galactosylceramide, etc.    -   A gamma inulin [107] or derivative thereof, such as algammulin.    -   An oil-in-water emulsion. Various such emulsions are known, and        they typically include at least one oil and at least one        surfactant, with the oil(s) and surfactant(s) being        biodegradable (metabolisable) and biocompatible. Further details        are given below.    -   An immunostimulatory oligonucleotide, such as one containing a        CpG motif (a dinucleotide sequence containing an unmethylated        cytosine residue linked by a phosphate bond to a guanosine        residue), or a CpI motif (a dinucleotide sequence containing        cytosine linked to inosine), or a double-stranded RNA, or an        oligonucleotide containing a palindromic sequence, or an        oligonucleotide containing a poly(dG) sequence.        Immunostimulatory oligonucleotides can include nucleotide        modifications/analogs such as phosphorothioate modifications and        can be double-stranded or (except for RNA) single-stranded.        References 108, 109 and 110 disclose possible analog        substitutions e.g. replacement of guanosine with        2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG        oligonucleotides is further discussed in refs. 111-116. A CpG        sequence may be directed to TLR9, such as the motif GTCGTT or        TTCGTT [117]. The CpG sequence may be specific for inducing a        Th1 immune response, such as a CpG-A ODN (oligodeoxynucleotide),        or it may be more specific for inducing a B cell response, such        a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in refs.        118-120. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG        oligonucleotide is constructed so that the 5′ end is accessible        for receptor recognition. Optionally, two CpG oligonucleotide        sequences may be attached at their 3′ ends to form “immunomers”.        See, for example, references 117 & 121-123. A useful CpG        adjuvant is CpG7909, also known as ProMune™ (Coley        Pharmaceutical Group, Inc.). Another is CpG1826. As an        alternative, or in addition, to using CpG sequences, TpG        sequences can be used [124], and these oligonucleotides may be        free from unmethylated CpG motifs. The immunostimulatory        oligonucleotide may be pyrimidine-rich. For example, it may        comprise more than one consecutive thymidine nucleotide (e.g.        TTTT, as disclosed in ref. 124), and/or it may have a nucleotide        composition with >25% thymidine        (e.g. >35%, >40%, >50%, >60%, >80%, etc.). For example, it may        comprise more than one consecutive cytosine nucleotide (e.g.        CCCC, as disclosed in ref. 124), and/or it may have a nucleotide        composition with >25% cytosine        (e.g. >35%, >40%, >50%, >60%, >80%, etc.). These        oligonucleotides may be free from unmethylated CpG motifs.        Immunostimulatory oligonucleotides will typically comprise at        least 20 nucleotides. They may comprise fewer than 100        nucleotides.    -   A particularly useful adjuvant based around immunostimulatory        oligonucleotides is known as IC31™ [125]. Thus an adjuvant used        with the invention may comprise a mixture of (i) an        oligonucleotide (e.g. between 15-40 nucleotides) including at        least one (and preferably multiple) CpI motifs, and (ii) a        polycationic polymer, such as an oligopeptide (e.g. between 5-20        amino acids) including at least one (and preferably multiple)        Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be a        deoxynucleotide comprising 26-mer sequence 5′-(IC)₁₃-3′ (SEQ ID        NO: ______). The polycationic polymer may be a peptide        comprising 11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO:        ______).    -   3-O-deacylated monophosphoryl lipid A (‘3dMPL’, also known as        ‘MPL™’) [126-129]. In aqueous conditions, 3dMPL can form        micellar aggregates or particles with different sizes e.g. with        a diameter <150 nm or >500 nm. Either or both of these can be        used with the invention, and the better particles can be        selected by routine assay. Smaller particles (e.g. small enough        to give a clear aqueous suspension of 3dMPL) are preferred for        use according to the invention because of their superior        activity [130]. Preferred particles have a mean diameter less        than 220 nm, more preferably less than 200 nm or less than 150        nm or less than 120 nm, and can even have a mean diameter less        than 100 nm. In most cases, however, the mean diameter will not        be lower than 50 nm.    -   An imidazoquinoline compound, such as Imiquimod (“R-837”)        [131,132], Resiquimod (“R-848”) [133], and their analogs; and        salts thereof (e.g. the hydrochloride salts). Further details        about immunostimulatory imidazoquinolines can be found in        references 134 to 138.    -   A thiosemicarbazone compound, such as those disclosed in        reference 139. Methods of formulating, manufacturing, and        screening for active compounds are also described in        reference 139. The thiosemicarbazones are particularly effective        in the stimulation of human peripheral blood mononuclear cells        for the production of cytokines, such as TNF-α.    -   A tryptanthrin compound, such as those disclosed in        reference 140. Methods of formulating, manufacturing, and        screening for active compounds are also described in        reference 140. The thiosemicarbazones are particularly effective        in the stimulation of human peripheral blood mononuclear cells        for the production of cytokines, such as TNF-α.    -   A nucleoside analog, such as: (a) Isatorabine (ANA-245;        7-thia-8-oxoguanosine).

-   -   and prodrugs thereof; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e)        the compounds disclosed in references 141 to 143Compounds        containing lipids linked to a phosphate-containing acyclic        backbone, such as the TLR4 antagonist E5564 [144,145]:

-   -   A substituted urea or compound of formula I, II or III, or a        salt thereof:

-   -   as defined in reference 146, such as ‘ER 803058’, ‘ER 803732’,        ‘ER 804053’, ER 804058′, ‘ER 804059’, ‘ER 804442’, ‘ER 804680’,        ‘ER 804764’, ER 803022 or ‘ER 804057’ e.g.:

-   -   Derivatives of lipid A from Escherichia coli such as OM-174        (described in refs. 147 & 148).    -   Loxoribine (7-allyl-8-oxoguanosine) [149].    -   Compounds disclosed in reference 150, including: Acylpiperazine        compounds, Indoledione compounds, Tetrahydraisoquinoline (THIQ)        compounds, Benzocyclodione compounds, Aminoazavinyl compounds,        Aminobenzimidazole quinolinone (ABIQ) compounds [151,152],        Hydrapthalamide compounds, Benzophenone compounds, Isoxazole        compounds, Sterol compounds, Quinazilinone compounds, Pyrrole        compounds [153], Anthraquinone compounds, Quinoxaline compounds,        Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole        compounds [154].    -   An aminoalkyl glucosaminide phosphate derivative, such as RC-529        [155,156].    -   A phosphazene, such as poly[di(carboxylatophenoxy)phosphazene]        (“PCPP”) as described, for example, in references 157 and 158.

These and other adjuvant-active substances are discussed in more detailin references 162 & 163.

Compositions may include two or more of said adjuvants. Individualadjuvants may preferentially induce either a Th1 response or a Th2response, and useful combinations of adjuvants can include both a Th2adjuvant (e.g. an oil-in-water emulsion or an aluminium salt) and a Th1adjuvant (e.g. 3dMPL, a saponin, or an immunostimulatoryoligonucleotide). For example, compositions may advantageously comprise:both an aluminium salt and an immunostimulatory oligodeoxynucleotide;both an aluminium salt and a compound of formula I, II or III; both anoil-in-water emulsion and a compound of formula I, II or III; both anoil-in-water emulsion and an immunostimulatory oligodeoxynucleotide;both an aluminium salt and an α-glycosylceramide; both an oil-in-wateremulsion and an α-glycosylceramide; both an oil-in-water emulsion and3dMPL; both an oil-in-water emulsion and a saponin; etc. Mixtures of3dMPL and oil-in-water emulsions are vey useful.

Preferred adjuvants for use with the invention are oil-in-wateremulsions, which have been found to be particularly suitable for use inadjuvanting influenza virus vaccines. Various such emulsions are known,and they typically include at least one oil and at least one surfactant,with the oil(s) and surfactant(s) being biodegradable (metabolizable)and biocompatible. The oil droplets in the emulsion are generally lessthan 5 μm in diameter, and ideally have a sub-micron diameter, withthese small sizes being achieved with a microfluidiser to provide stableemulsions. Droplets with a size less than 220 nm are preferred as theycan be subjected to filter sterilization.

The emulsion can comprise oils such as those from an animal (such asfish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g. obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoids known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also a preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Other preferred oils are thetocopherols (see below). Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear ED/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are Tween 80(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate),lecithin and Triton X-100.

Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Preferred emulsion adjuvants have an average droplets size of <1 μm e.g.≦750 nm, ≦500 nm, ≦400 nm, ≦300 nm, ≦250 nm, ≦220 nm, ≦200 nm, orsmaller. These droplet sizes can conveniently be achieved by techniquessuch as microfluidisation.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, polysorbate 80, and sorbitan        trioleate. The composition of the emulsion by volume can be        about 5% squalene, about 0.5% polysorbate 80 and about 0.5%        sorbitan trioleate. In weight terms, these ratios become 4.3%        squalene, 0.5% polysorbate 80 and 0.48% sorbitan trioleate. This        adjuvant is known as ‘MF59’ [159-161], as described in more        detail in Chapter 10 of ref. 162 and chapter 12 of ref. 163. The        MF59 emulsion advantageously includes citrate ions e.g. 10 mM        sodium citrate buffer.    -   An emulsion of squalene, a tocopherol, and polysorbate 80. The        emulsion may include phosphate buffered saline. It may also        include Span 85 (e.g. at 1%) and/or lecithin. These emulsions        may have from 2 to 10% squalene, from 2 to 10% tocopherol and        from 0.3 to 3% polysorbate 80, and the weight ratio of        squalene:tocopherol is preferably ≦1 as this provides a more        stable emulsion. Squalene and Tween 80 may be present volume        ratio of about 5:2 or at a weight ratio of about 11:5. One such        emulsion can be made by dissolving Tween 80 in PBS to give a 2%        solution, then mixing 90 ml of this solution with a mixture of        (5 g of DL-α-tocopherol and 5 ml squalene), then microfluidising        the mixture. The resulting emulsion may have submicron oil        droplets e.g. with an average diameter of between 100 and 250        nm, preferably about 180 nm. The emulsion may also include a        3-de-O-acylated monophosphoryl lipid A (3d-MPL). Another useful        emulsion of this type may comprise, per human dose, 0.5-10 mg        squalene, 0.5-11 mg tocopherol, and 0.1-4 mg polysorbate 80        [164].    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see        below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“Pluronic™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant [165] (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant [166]        (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).        Microfluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [167]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. The emulsion may        include a TLR4 agonist [168]. Such emulsions may be lyophilized.    -   An emulsion of squalene, poloxamer 105 and Abil-Care [169]. The        final concentration (weight) of these components in adjuvanted        vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and        2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone;        caprylic/capric triglyceride).    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 170, preferred phospholipid components        are phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   A submicron oil-in-water emulsion of a non-metabolisable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, Tween 80 or Span 80). Additives may be included, such        as QuilA saponin, cholesterol, a saponin-lipophile conjugate        (such as GPI-0100, described in reference 171, produced by        addition of aliphatic amine to desacylsaponin via the carboxyl        group of glucuronic acid), dimethyldioctadecylammonium bromide        and/or N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine.    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [172].    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [173].    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [173].

In some embodiments an emulsion may be mixed with antigenextemporaneously, at the time of delivery, and thus the adjuvant andantigen may be kept separately in a packaged or distributed vaccine,ready for final formulation at the time of use. In other embodiments anemulsion is mixed with antigen during manufacture, and thus thecomposition is packaged in a liquid adjuvanted form, as in the FLUAD™product. The antigen will generally be in an aqueous form, such that thevaccine is finally prepared by mixing two liquids. The volume ratio ofthe two liquids for mixing can vary (e.g. between 5:1 and 1:5) but isgenerally about 1:1. Where concentrations of components are given in theabove descriptions of specific emulsions, these concentrations aretypically for an undiluted composition, and the concentration aftermixing with an antigen solution will thus decrease.

After the antigen and adjuvant have been mixed, haemagglutinin antigenwill generally remain in aqueous solution but may distribute itselfaround the oil/water interface. In general, little if any haemagglutininwill enter the oil phase of the emulsion.

Where a composition includes a tocopherol, any of the α, β, γ, δ, ε or ξtocopherols can be used, but α-tocopherols are preferred. The tocopherolcan take several forms e.g. different salts and/or isomers.

Salts include organic salts, such as succinate, acetate, nicotinate,etc. D-α-tocopherol and DL-α-tocopherol can both be used. Tocopherolsare advantageously included in vaccines for use in elderly patients(e.g. aged 60 years or older) because vitamin E has been reported tohave a positive effect on the immune response in this patient group[174]. They also have antioxidant properties that may help to stabilizethe emulsions [175]. A preferred α-tocopherol is DL-α-tocopherol, andthe preferred salt of this tocopherol is the succinate. The succinatesalt has been found to cooperate with TNF-related ligands in vivo.Moreover, α-tocopherol succinate is known to be compatible withinfluenza vaccines and to be a useful preservative as an alternative tomercurial compounds [28].

The Child

The invention is used to immunize children against influenza virusinfection and/or disease.

The child to be immunized may be aged between 0 months and 72 months,and ideally between 0 months and 36 months. Typically they will be atleast 6 months old e.g. in the range 6-72 months old (inclusive) or inthe range 6-36 months old (inclusive). Children in these age ranges mayin some embodiments be less than 30 months old, or less than 24 monthsold. For example, a composition may be administered to them at the ageof 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 months; or at 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71 months; orat 36 or 72 months.

Pharmaceutical Compositions

Compositions of the invention are pharmaceutically acceptable. They mayinclude components in addition to the antigen and adjuvant e.g. theywill typically include one or more pharmaceutical carrier(s) and/orexcipient(s). A thorough discussion of such components is available inref. 176.

The composition may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e. less than 5 μg/ml) mercurial material e.g.thiomersal-free [177,178]. Vaccines containing no mercury are morepreferred, and α-tocopherol succinate can be included as an alternativeto mercurial compounds [28]. Preservative-free vaccines are mostpreferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [179], but keeping osmolality in this range is neverthelesspreferred.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer (particularly with an aluminum hydroxide adjuvant); ora citrate buffer. Buffers will typically be included in the 5-20 mMrange.

The pH of a composition will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.A process of the invention may therefore include a step of adjusting thepH of the bulk vaccine prior to packaging.

The composition is preferably sterile. The composition is preferablynon-pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose. The composition is preferablygluten free.

Compositions of the invention may include detergent e.g. apolyoxyethylene sorbitan ester surfactant (known as ‘Tweens’), anoctoxynol (such as octoxynol-9 (Triton X-100) ort-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide(‘CTAB’), or sodium deoxycholate, particularly for a split or surfaceantigen vaccine. The detergent may be present only at trace amounts.Thus the vaccine may included less than 1 mg/ml of each of octoxynol-10and polysorbate 80. Other residual components in trace amounts could beantibiotics (e.g. neomycin, kanamycin, polymyxin B).

The composition may include material for a single immunisation, or mayinclude material for multiple immunisations (i.e. a ‘multidose’ kit).The inclusion of a preservative is preferred in multidose arrangements.As an alternative (or in addition) to including a preservative inmultidose compositions, the compositions may be contained in a containerhaving an aseptic adaptor for removal of material.

Influenza vaccines are typically administered in a dosage volume ofabout 0.5 ml, although a half dose (i.e. about 0.25 ml) may beadministered to children according to the invention.

Compositions and kits are preferably stored at between 2° C. and 8° C.They should not be frozen. They should ideally be kept out of directlight.

The antigen and emulsion in a composition will typically be inadmixture, although they may initially be presented in the form of a kitof separate components for extemporaneous admixing. Compositions willgenerally be in aqueous form when administered to a subject.

Kits of the Invention

Compositions of the invention may be prepared extemporaneously, at thetime of delivery, particularly when an adjuvant is being used. Thus theinvention provides kits including the various components ready formixing. The kit allows the adjuvant and the antigen to be keptseparately until the time of use. This arrangement can be useful whenusing an oil-in-water emulsion adjuvant.

The components are physically separate from each other within the kit,and this separation can be achieved in various ways. For instance, thetwo components may be in two separate containers, such as vials. Thecontents of the two vials can then be mixed e.g. by removing thecontents of one vial and adding them to the other vial, or by separatelyremoving the contents of both vials and mixing them in a thirdcontainer.

In a preferred arrangement, one of the kit components is in a syringeand the other is in a container such as a vial. The syringe can be used(e.g. with a needle) to insert its contents into the second containerfor mixing, and the mixture can then be withdrawn into the syringe. Themixed contents of the syringe can then be administered to a patient,typically through a new sterile needle. Packing one component in asyringe eliminates the need for using a separate syringe for patientadministration.

In another preferred arrangement, the two kit components are heldtogether but separately in the same syringe e.g. a dual-chamber syringe,such as those disclosed in references 180-187 etc. When the syringe isactuated (e.g. during administration to a patient) then the contents ofthe two chambers are mixed. This arrangement avoids the need for aseparate mixing step at the time of use.

The kit components will generally be in aqueous form. In somearrangements, a component (typically an antigen component rather than anadjuvant component) is in dry form (e.g. in a lyophilised form), withthe other component being in aqueous form. The two components can bemixed in order to reactivate the dry component and give an aqueouscomposition for administration to a patient. A lyophilised componentwill typically be located within a vial rather than a syringe. Driedcomponents may include stabilizers such as lactose, sucrose or mannitol,as well as mixtures thereof e.g. lactose/sucrose mixtures,sucrose/mannitol mixtures, etc. One possible arrangement uses an aqueousadjuvant component in a pre-filled syringe and a lyophilised antigencomponent in a vial.

Packaging of Compositions or Kit Components

Suitable containers for compositions of the invention (or kitcomponents) include vials, syringes (e.g. disposable syringes), nasalsprays, etc. These containers should be sterile.

Where a composition/component is located in a vial, the vial ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials are preferably sealed with a latex-freestopper, and the absence of latex in all packaging material ispreferred. The vial may include a single dose of vaccine, or it mayinclude more than one dose (a ‘multidose’ vial) e.g. 10 doses. Preferredvials are made of colorless glass.

A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g. to reconstitute lyophilised materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed. A vial may havea cap that permits aseptic removal of its contents, particularly formultidose vials.

Where a component is packaged into a syringe, the syringe may have aneedle attached to it. If a needle is not attached, a separate needlemay be supplied with the syringe for assembly and use. Such a needle maybe sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch25-gauge and 5/8-inch 25-gauge needles are typical. Syringes may beprovided with peel-off labels on which the lot number, influenza seasonand expiration date of the contents may be printed, to facilitate recordkeeping. The plunger in the syringe preferably has a stopper to preventthe plunger from being accidentally removed during aspiration. Thesyringes may have a latex rubber cap and/or plunger. Disposable syringescontain a single dose of vaccine. The syringe will generally have a tipcap to seal the tip prior to attachment of a needle, and the tip cap ispreferably made of a butyl rubber. If the syringe and needle arepackaged separately then the needle is preferably fitted with a butylrubber shield. Useful syringes are those marketed under the trade name“Tip-Lok”™.

Containers may be marked to show a half-dose volume e.g. to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

A kit or composition may be packaged (e.g. in the same box) with aleaflet including details of the vaccine e.g. instructions foradministration, details of the antigens within the vaccine, etc. Theinstructions may also contain warnings e.g. to keep a solution ofadrenaline readily available in case of anaphylactic reaction followingvaccination, etc.

Methods of Treatment, and Administration of the Vaccine

Compositions of the invention are suitable for administration to humanpatients, and the invention provides a method of raising an immuneresponse in a patient, comprising the step of administering acomposition of the invention to the patient. As described above, thepatient is a child.

The invention also provides a kit or composition of the invention foruse as a medicament. The invention also provides the medical usesdiscussed above.

These methods and uses will generally be used to generate an antibodyresponse, preferably a protective antibody response. Methods forassessing antibody responses, neutralising capability and protectionafter influenza virus vaccination are well known in the art. Humanstudies have shown that antibody titers against hemagglutinin of humaninfluenza virus are correlated with protection (a serum samplehemagglutination-inhibition titer of about 30-40 gives around 50%protection from infection by a homologous virus) [188]. Antibodyresponses are typically measured by hemagglutination inhibition (HI), bymicroneutralisation (Micro-NT), by single radial immunodiffusion (SRID),and/or by single radial hemolysis (SRH). These assay techniques are wellknown in the art.

Compositions of the invention can be administered in various ways. Themost preferred immunisation route is by intramuscular injection (e.g.into the arm or leg), but other available routes include subcutaneousinjection, intranasal [189-191], oral [192], intradermal [193,194],transcutaneous, transdermal [195], etc.

Preferred compositions of the invention will satisfy 1, 2 or 3 of theCPMP criteria for adult efficacy for each influenza strain, even thoughthey are administered to children. These criteria are: (1)≧70%seroprotection; (2)≧40% seroconversion or significant increase; and/or(3) a GMT increase of ≧2.5-fold. In elderly (≧60 years), these criteriaare: (1)≧60% seroprotection; (2)≧30% seroconversion; and/or (3) a GMTincrease of ≧2-fold. These criteria are based on open label studies withat least 50 patients.

The invention is particularly useful for raising immune responses thatare protective against influenza B virus strains and/or are effectiveagainst drifted (mismatched) influenza A virus strains (particularlydrifted A/H3N2 strains).

Treatment can be by a single dose schedule or a multiple dose schedule.In any particular influenza season (e.g. in a given 12 month period,typically in autumn or winter) a patient may thus receive a single doseof a composition of the invention or more than one dose (e.g. twodoses). A single dose can raise a useful immune response against subtypeH3N2 of influenza A virus, whereas two doses may be required toadditionally provide a useful immune response against subtype H1N1 ofinfluenza A virus and against influenza B virus. In a multiple doseschedule the various doses may be given by the same or different routese.g. a parenteral prime and mucosal boost, a mucosal prime andparenteral boost, etc. Typically they will be given by the same route.Multiple doses will typically be administered at least 1 week apart(e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 12 weeks, about 16 weeks, etc.). Giving two dosesseparated by from 25-30 days (e.g. 28 days) is particularly useful.

Vaccines produced by the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines e.g. at substantially the same time as a measlesvaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicellavaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a DTP vaccine, a conjugated H. influenzae type bvaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine,a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Yvaccine), a pneumococcal conjugate vaccine, etc.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g. oseltamivir and/or zanamivir). These antiviralsinclude neuraminidase inhibitors, such as a(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate(TAMIFLU™).

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means, for example,x+10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

Where a cell substrate is used for reassortment or reverse geneticsprocedures, it is preferably one that has been approved for use in humanvaccine production e.g. as in Ph Eur general chapter 5.2.3.

MODES FOR CARRYING OUT THE INVENTION The Clinical Trial

A phase II clinical trial has been performed in children to assess theimmunogenicity, clinical tolerability and safety of an adjuvantedinactivated influenza vaccine in comparison to a non-adjuvantedinactivated vaccine in unprimed healthy children.

Healthy children (6 to <36 months of age) never being previouslyvaccinated against influenza were invited to participate in the trial.To ensure equal age distribution within this age range, the followingsubgroups of children were targeted for recruitment: 6-11 months, 12-18months, 19-24 months, 24-30 months, 31-<36 months. Subjects wererandomized to receive one of the two trivalent inactivated influenzavaccines: a subunit vaccine adjuvanted with MF59™ (FLUAD™), or anon-adjuvanted split vaccine (Vaxigrip™). Two doses, 0.25 ml each, weregiven intramuscularly in the deltoid region of the non-dominant arm or,if the deltoid mass was insufficient, in the anterolateral aspect of thethigh. The second vaccination was four weeks after the first.

The antigenic composition of the two vaccines was in agreement with WHOrecommendations for the Northern Hemisphere during the 2006/07 influenzaseason. For each dose of 0.25 ml vaccines contained 7.5 μg of each ofthe three influenza antigens: A/New Calcdonia/20/99 (H1N1)-like virus,A/Wisconsin/67/2005 (H3N2)-like virus, B/Malaysia/2506/2004-like virus.

The study protocol conformed to the ethical guidelines of the 1975Declaration of Helsinki and Good Clinical Practice and was approved bythe Ethics Committee of the University Hospital District of Tampere.Counseling was provided and informed consent was obtained from eachchild's parents. Exclusion criteria included subjects who had a knownallergy to any vaccine component or had experienced any known orsuspected neurological reactions following influenza vaccination; hadexperienced any acute infectious or respiratory disease requiringsystemic treatment 30 days before the study start; had experienced alaboratory confirmed influenza disease in the previous 6 months. Foreach child, a detailed form including demographic and baseline clinicaldata was completed. Blood samples were obtained before vaccination, fourweeks after the first vaccine dose and three weeks after the second one.A fourth blood draw was performed at the study termination, at the endof the six months follow up, to assess immune responses over theduration of an influenza season.

H1 antibody titres were measured in all samples against each of thethree influenza strains in the vaccine formulation. The following immuneparameters were considered: Geometric Mean Titres (GMT) and thecorresponding 95% confidence intervals (CI); Geometric Mean Ratio (GMR;ratio of post- to pre-vaccination titre); seroprotection rate, definedas the percentage of subjects achieving an HI titre ≧40; and thepercentage of subjects achieving seroconversion (defined as at least a4-fold increase in HI titre from a non-negative pre-vaccination titre[≧10] or a rise from <10 to ≧40 in those who were seronegative). Inaddition, the percentage of subjects with HI titres ≧160 was evaluated.

Immediately after any vaccination and for the following seven days,parents were instructed to record solicited local and systemic reactionson a diary card. Body temperature, usage of analgesic/antipyreticmedication and any other adverse event were registered on diary cardsbeginning with the day of vaccination and continuing during the 7 daysfollowing each vaccination.

All adverse events (AE) including serious adverse events and thosenecessitating a physician's consultation, or leading to premature studydiscontinuation were collected throughout the entire trial.

Data were statistically analysed using the SAS System. The chi-squaretest was performed to analyze differences between proportions ofsubjects. Statistical significance between pre- and post-vaccinationtitres was calculated using the paired Student's t-test. Comparison ofdifferent vaccine groups was determined by Student's t-test for unpaireddata. A P-value of <0.05 was considered to indicate statisticalsignificance.

Results are shown in Table I (after 1 dose) and Table II (after 2doses). The seroprotection rate is the percentage of children achievingan HI titre ≧40. The seroconversion rate is the percentage of subjectsachieving seroconversion or a significant increase in titre (i.e. atleast a 4-fold increase in HI titre from a non-negative pre-vaccinationtitre [≧10] or a rise from <10 to ≧40 in those who were serum-negative).Statistical significance is indicated vs. un-adjuvanted group as:*p<0.001; **p<0.01; ***p<0.05.

Safety

Overall, 269 children were enrolled and randomized into five age groupsto receive the vaccines. Diary cards for local and systemic reactionsand AE reports were collected from all 269 children.

There was no statistically significant difference in local and systemicreactions between the two vaccine groups, with the only exception ofinjection site swelling. All reactions were typically mild or moderateand transient (2-3 days after vaccination). In general, after the secondvaccine dose, administered four weeks apart, the trend was for areduction both in local and systemic reactions recorded, compared to thefirst vaccination.

The overall analysis of possibly or probably related adverse events,from the study start to end of the six months follow up, found nodifferences between vaccine groups (21 children in each group, with nosevere AE reported). Two children in each group were withdrawn from thetrial because of an AE. Two serious adverse events (SAE) were reportedduring the follow up period in the adjuvanted group (two cases ofpneumonia); six SAE were recorded in the unadjuvanted group (two chronicbronchitis, two cases of gastroenteritis, one otitis media and one caseof asthma). None of them was judged vaccine-related.

Immunogenicity

Serological analysis was performed on the 222 subjects who completed thefull vaccination schedule and had all four sera drawn. The distributionto age subgroups was well matched between groups.

By all comparisons, the immune responses to adjuvanted vaccine weresuperior to those after unadjuvanted vaccine. Thus adjuvanted influenzavaccines may become the preferred influenza vaccine for young childrenaged 6 to <36 months.

Baseline GMTs were well balanced between vaccine groups. By allcomparisons, immune responses were strongest against H3N2, followed byH1N1 and B, and are presented in this order. The GMTs three weeks after2nd vaccine dose against H3N2 strain were significantly higher thanthose recorded versus H1N1 antigen, which in turn were significantlyhigher than GMTs to influenza B. Furthermore GMTs against H3N2, H1N1 andB, respectively, were significantly higher after vaccination withadjuvanted vaccine than after unadjuvanted vaccine (all comparisonsp<0.001).

The same trend was observed for immunogenicity results six months aftervaccine schedule completion, confirming higher antibody persistence inchildren vaccinated with adjuvanted vaccine. Although titers declinedover this six month period, they were consistently higher in therecipients of adjuvanted vaccine than in recipients of the unadjuvantedvaccine.

Both vaccines yielded high seroprotection rates against H3N2 after twodoses of vaccine (100% for adjuvanted vaccine and 99% for unadjuvantedvaccine). However, after one dose, there was a considerable andsignificant difference in favor of the adjuvanted vaccine, as 91% of theadjuvanted vaccine recipients reached seroprotection level already atthis point, compared with only 49% of the unadjuvanted vaccinerecipients (p<0.001). After six months the seroprotection rate for theadjuvanted vaccine remained at 100%, against 66% for the unadjuvantedvaccine (p<0.001), and so the adjuvanted vaccine can offer sustainedimmune protection throughout an influenza season.

Against H1N1, two doses of adjuvanted vaccine also resulted in 100%seroprotection rate vs. 86% after unadjuvanted vaccine (p<0.001). Afterone dose, the adjuvanted vaccine yielded 51% seroprotection rate vs.only 18% in the unadjuvanted vaccine group (p<0.001). After six monthsthe seroprotection rate with the adjuvanted vaccine was againsignificantly higher (p<0.001) than with the unadjuvanted vaccine.

Antibody responses to influenza B were weak after one dose, but aftertwo doses of adjuvanted vaccine 99% of the recipients had seroprotectivelevel of H1 antibody vs. only 33% of the recipients of the unadjuvantedvaccine. The lower immunogenicity of influenza B is in accordance withprevious studies [13,14]. After six months, the seroprotection rate withthe adjuvanted vaccine was again significantly higher (p<0.001) thanwith the unadjuvanted vaccine.

The HI antibody responses against A/H1N1 and A/H3N2 strains after eachvaccine were essentially similar in the youngest subgroups i.e. therewas no apparent improvement with increasing age, whereas for the Bstrain a consistent trend of decreasing antibody response was observedin the lower age groups vaccinated with the unadjuvanted vaccine group.This might indicate that the youngest children are responding less tounadjuvanted vaccines even after the second dose, compared to anadjuvanted vaccine. Conversely, the high antibody response to theadjuvanted vaccine could already be seen in the youngest infants, 6 to11 months old.

In practice, despite recommendations, young children often only receiveone injection of influenza vaccine in a season. Therefore, a vaccineable to elicit higher antibody titers after the first dose may result inimproved field efficacy in children. The present study indicated that,for conventional unadjuvanted vaccine, a single dose is not sufficientto induce protective immunity against H1N1 influenza A virus orinfluenza B virus, and is clearly suboptimal for H3N2 as well. A/H3N2 iscurrently the most common circulating influenza virus strain, and theadjuvanted vaccine yielded high seroprotection (91%) against this straineven after one dose. FIG. 4 shows that significantly (P<0.001) higherseroprotection rates were obtained with the adjuvanted vaccine forA/H3N2 and A/H1N1 following dose 1, and that this difference wasmaintained until the end of the study (Day 209). Importantly, theresponse to H3N2 was also more durable in subjects in the adjuvantedgroup, with 88% of subjects retaining seroprotective levels of antibodyone year after their first influenza vaccination.

Immune Responses Versus Mismatched Strains

As the study used previously-unvaccinated healthy children, thecross-protection potential achieved by the inclusion of adjuvant in thevaccine was directly compared to immunization with a conventionalunadjuvanted influenza vaccine. The recent recommendations for strainchanges for all three seasonal strains in the vaccine created anopportunity to broadly assess sera from vaccine-immunized subjects forcross reactivity against natural drift strains of influenza virus.

Subjects were immunized with the vaccine recommended for the 2006/07season, and sera were evaluated against the A/H3N2 and B strains thathad been included in the 2005/06 vaccine, before the drift occurred. Inaddition, heterologous activity was measured against a drifted A/H1N1strain which was recommended for inclusion in the 2007/08 season. Hence,the changes in all three vaccine strains which were recommended over atwo year period offered a unique opportunity to assess thecross-immunogenicity potential achieved by the inclusion of adjuvant inthis vaccine-naïve population of young children.

Cross-immunogenicity against mismatched influenza strains was thusevaluated using sera from the children.

Results are in Table III (the * indicates P<0.001).

For both vaccine groups, pre-vaccination GMTs and seroprotection rateswere higher for the heterovariant A/H3N2 virus strain than for the othertwo (A/H1N1 and B) heterologous antigens. Both vaccines induced asignificant rise (P<0.001) in GMTs against drifted influenza strains at3 weeks post-vaccination.

For all three strains, significantly higher GMTs (P<0.001) were recordedin the adjuvanted group, compared to the unadjuvanted group.Furthermore, significantly higher (P<0.001) GMRs were detected in theadjuvanted vaccine group, compared to the unadjuvanted group (A/H3N2: 13vs. 4.78; A/H1N1: 9.11 vs. 4; B: 2.12 vs. 1.21, respectively).

Satisfactory post-vaccination seroprotection and seroconversion rateswere reached in the adjuvanted group against both mismatched A influenzastrains in the MF59 group, but not for the B drifted strain. Thedifferences between vaccine groups were statistically significant forall three strains for seroprotection rates, but only for A strains forseroconversion rates.

The analysis of the immunogenicity results according to the Committeefor Medicinal Products for Human Use (CHMP; formerly CPMP) criteria foryearly approval of licensed influenza vaccines in healthy adults showedthat all 3 criteria were fulfilled for both A antigens in the adjuvantedgroup, while the unadjuvanted vaccine met only 2 requirements (GMR andseroconversion rate) for the A/H3N2 and only the 1 (mean fold increasein titers) for the A/H1N1 strain.

Thus the inclusion of adjuvant in the influenza vaccine alloweddivergent strains, which were sufficiently different to result in achange in recommendation for vaccine strains, to be covered by aprotective serum immune response. The drift cover was significantlyhigher than achieved with an unadjuvanted vaccine. A publishedmulti-year study [196] which evaluated influenza vaccine effectivenessversus antigenic distance of strain mismatches in the vaccine suggeststhat the cross reactivity achieved with the adjuvanted vaccine wouldlikely have a significant clinical impact.

Observer-Blind Extension Study

Children who had been primed in the initial clinical study were offeredto receive a booster dose of the adjuvanted vaccine or unadjuvantedsplit vaccine one year later. Healthy children (now aged 16 to <48months) who had been primed with two intramusluclar (IM) doses for the2006/07 season thus received a third intramuscular dose of therespective vaccine (2007/08 NH vaccine formulation) approximately oneyear after the first dose (before the start of the 2007/08 season). Forthe 2007/08 NH season only the A/H1N1-like strain (A/SolomonIslands/3/2006) changed compared with the vaccine formulation of theprevious campaign. The third dose of the vaccines was thus like a“booster dose” for the A/H3N2 and B strains, which did not change acrossthe two seasons.

Immunogenicity was evaluated by a haemagglutination inhibition (HI)assay at baseline, before the booster dose, and three weeks after.Seroprotection (SP) was defined as HI titer of 40 or higher andseroconversion (SC) was defined as a ≧4-fold increase in HI titre from apre-vaccination titre ≧10 or a rise from <10 to ≧40. Solicited local andsystemic reactions were monitored immediately after vaccination and forthe following seven days. All adverse events (AE) were recorded up to 3weeks after injection.

Overall, 89 children took part in this extension study. Both vaccineswere confirmed to be safe and well tolerated after a second seasonalvaccination. Mild solicited reactions were more frequently recorded inthe adjuvanted group, whereas AEs were more common in the split group.

Baseline HI antibody titers, SP rates and SC rates were higher in theadjuvanted group compared with the split group. The difference inpersistence of antibody titers, approximately one year after priming,was particularly evident against the A/H3N2 strain (adjuvanted 88% SPvs. unadjuvanted 40% SP, p<0.001). For both vaccines the immuneresponses after vaccination were strongest against A/H3N2, followed byA/H1N1 and B. The adjuvanted vaccine induced significantly higher GMTsthan the unadjuvanted vaccine against all three vaccine strains. Allsubjects in the adjuvanted group achieved SP against all three vaccinestrains whereas split vaccine conferred seroprotection in the 68% ofchildren against the B antigen (p<0.001). The same trend was observedfor the percentage of children achieving SC, with the greatestdifference between groups being for the B strain (98% in the adjuvantedgroup vs. 68% in the unadjuvanted group, p<0.001). Results are in TableIV.

After the second year booster dose, seroprotection against the B strainin younger children remained at less than 50% in the unadjuvantedvaccine group compared with 100% in the adjuvanted group.

Thus the adjuvanted influenza vaccine was confirmed to be safe and welltolerated following a second consecutive seasonal vaccination. BaselineHI antibody titers, were consistently higher in children receivingadjuvanted vaccine, confirming a better persistence of immunogenicityafter priming than with a conventional vaccine. The adjuvanted vaccineinduced higher increases in immune responses three weeks aftervaccination, especially in the youngest children (<3 years of age) andagainst the B influenza strain, which is epidemiologically relevant inthe pediatric population. These data further support the use ofadjuvanted vaccine as a safe and very immunogenic influenza vaccine forchildren.

The results of this extension study, performed to mimic the ideal fieldconditions of consecutive seasonal vaccinations, further support the useof adjuvanted vaccines as a highly immunogenic and well-tolerated wayfor actively immunizing against seasonal influenza in healthy children.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

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TABLE I Age Groups up to 11 months 12-17 months 18-23 months 24-29months 30-35 months Overall Population Immunogenicity Adjuv UnadjuvAdjuv Unadjuv Adjuv Unadjuv Adjuv Unadjuv Adjuv Unadjuv Adjuv UnadjuvStrain Endpoints N = 19 N = 26 N = 22 N = 15 N = 22 N = 26 N = 18 N = 30N = 23 N = 21 N = 104 N = 118 A/H3N2 GMT 77** 27 88** 28 94 48 145** 30111 71 100* 38 (95% CI) (45-131) (17-42) (56-138) (16-48) (48-183)(26-89) (67-313) (17-55) (48-261) (29-174) (74-135) (28-50) GMR 15* 3.7914** 5.53 11** 4.69 11* 4.09 11* 3.93 12* 4.28 (95% CI) (11-22)(2.80-5.13) (9.56-20) (3.59-8.52) (6.95-16) (3.18-6.93) (7.55-15)(3.13-5.35) (7.98-16) (2.77-5.58) (10-14) (3.69-4.97) Seroprotection 95*38 91*** 53 91*** 62 89** 47 91** 48 91* 49 % (95% CI) (74-100) (20-59)(71-99) (27-79) (71-99) (41-80) (65-99) (28-66) (72-99) (26-70) (84-96)(40-59) HI titre ≧ 160 16 8 27 0 27 19 33 10 22 33 25*** 14 % (3-40)(1-25) (11-50) (0-22) (11-50) (7-39) (13-59) (2-27) (7-44) (15-57)(17-34) (9-22) (95% CI) Seroconversion 95* 35 86 53 86*** 54 89** 47 91*38 89* 45 % (74-100) (17-56) (65-97) (27-79) (65-97) (33-73) (65-99)(28-66) (72-99) (18-62) (82-95) (36-54) (95% CI) A/H1N1 GMT 30*** 1327** 14 25 27 48** 12 47 25 34* 17 (95% CI) (18-49) (8.62-20) (20-36)(9.40-19) (12-50) (14-51) (25-91) (7.04-19) (23-95) (12-52) (26-44)(13-21) GMR 5.98* 2.29 5.31** 2.70 4.99*** 2.90 6.60* 2.02 5.74 3.815.66* 2.61 (95% CI) (4.28-8.35) (1.72-3.04) (3.94-7.16) (1.88-3.88)(3.52-7.06) (2.11-4.00) (4.57-9.53) (1.52-2.69) (3.90-8.46) (2.54-5.71)(4.85-6.60) (2.26-3.02) Seroprotection 47*** 15 41 13 45 (24-68) 19 56**13 65*** 29 51* 18 % (24-71%) (4-35%) (21-64%) (2-40) (7-39) (31-78)(4-31) (43-84) (11-52) (41-61) (11-26) (95% CI) HI titre ≧ 160 0 4 0 0 015 11 3 13 10 5 7 % (95% CI) (0-18) (0.097-20) (0-15) (0-22) (0-15)(4-35) (1-35) (0.084-17) (3-34) (1-30) (2-11) (3-13) Seroconversion47*** 15 41 13 45*** 15 56** 13 65*** 29 51* 17 % (95% CI) (24-71)(4-35) (21-64) (2-40) (24-68) (4-35) (31-78) (4-31) (43-84) (11-52)(41-61) (11-25) B GMT 7.47 5.00 7.30*** 5.24 5.33 6.36 15*** 5.74 8.866.73 8.11** 5.79 (95% CI) (5.10-11) (3.61-6.92) (6.02-8.85) (4.15-6.61)(3.86-7.34) (4.73-8.54) (8.01-28) (3.54-9.33) (5.78-14) (4.30-11)(6.75-9.74) (4.87-6.88) GMR 1.29 1.00 1.46*** 1.05 1.07 1.21 2.47***1.12 1.62 1.22 1.50** 1.12 (95% CI) (1.06-1.57) (0.84-1.18) (1.20-1.77)(0.83-1.32) (0.83-1.36) (0.96-1.51) (1.48-4.13) (0.75-1.67) (1.23-2.13)(0.91-1.62) (1.31-1.72) (0.98-1.27) Seroprotection 5 0 0 0 0 4 17 3 4 55 3 % (0-26) (0-13) (0-15) (0-22) (0-15) (0.097-20) (4-41) (0.084-17)(0-22) (0-24) (2-11) (1-7) (95% CI) HI titre ≧ 160 5 0 0 0 0 4 17 3 4 55 3 % (0-26) (0-13) (0-15) (0-22) (0-15) (0.097-20) (4-41) (0.084-17)(0-22) (0-24) (2-11) (1-7) (95% CI) Seroconversion 5 0 0 0 0 4 17 3 4 55 3 % (95% CI) (0-26%) (0-13%) (0-15%) (0-22%) (0-15%) (0.097-20%)(4-41%) (0.084-17%) (0-22%) (0-24%) (2-11%) (1-7%)

TABLE II Age Groups up to 11 months 12-17 months 18-23 months 24-29months 30-35 months Overall Population Immunogenicity Split UnadjuvAdjuv Unadjuv Adjuv Unadjuv Adjuv Unadjuv Unadjuv Strain Endpoints AdjuvN = 19 N = 26 Adjuv N = 22 N = 15 N = 22 N = 26 N = 18 N = 30 N = 23 N =21 Adjuv N = 104 N = 118 A/H3N2 GMT 514* 135 381** 156 521*** 259 518*168 630 315 507* 195 (95% CI) (326-811) (91-199) (263-531) (100-245)(330-824) (170-394) (324-828) (116-241) (362-1098) (176-563) (412-623)(160-237) GMR 103* 19 59*** 31 59*** 25 38 23 63* 17 61* 22 (95% CI)(66-160) (13-28) (40-87) (20-50) (35-99) (16-40) (23-62) (16-33) (40-99)(11-28) (50-75) (18-27) Seroprotection^(a) 100 96 100 100 100 100 100100 100 100 100 99 % (82-100) (80-100) (85-100) (78-100) (85-100)(87-100) (81-100) (88-100) (85-100) (84-100) (97-100) (95-100) (95% CI)HI titre ≧ 160 100* 54 95*** 67 95** 62 100** 70 100** 71 98* 64 % (95%CI) (82-100) (33-73) (77-100) (38-88) (77-100) (41-80) (81-100) (51-85)(85-100) (48-89) (93-100) (55-73) Seroconversion^(b) 100 92 100 100 9596 94 100 100 90 98 96 % (95% CI) (82-100) (75-99) (85-100) (78-100)(77-100) (80-100) (73-100) (88-100) (85-100) (70-99) (93-100) (90-99)A/H1N1 GMT 218* 76 163** 80 165 123 205* 69 240 133 195* 92 (95% CI)(144-332) (53-108) (120-221) (55-116) (97-282) (75-201) (134-316)(49-96) (137-421) (74-240) (159-240) (76-111) GMR 44* 13 33** 16 33* 1329* 12 30 20 33* 14 (95% CI) (30-63) (9.69-18) (22-44) (11-23) (23-47)(9.68-18) (21-39) (9.39-15) (20-44) (13-31) (28-38) (12-17)Seroprotection^(a) 100 85 100 100 100 88 100 83 100*** 81 100* 86 %(82-100) (65-96) (85-100) (78-100) (85-100) (70-98) (87-100) (65-94)(85-100) (58-95) (97-100) (79-92) (95% CI) HI titre ≧ 160 79* 27 68** 2764*** 35 72* 17 70 52 70* 31 % (95% CI) (54-94) (12-48) (45-86) (8-55)(41-83) (17-56) (47-90) (6-35) (47-87) (30-74) (60-79) (22-40)Seroconversion^(b) 100 85 100 100 100 85 100 83 100*** 81 100* 86 %(82-100) (65-96) (85-100) (78-100) (85-100) (65-96) (82-100) (65-94)(85-100) (58-95) (97-100) (78-91) (95% CI) B GMT 96* 11 95* 19 80* 24129* 23 140* 33 105* 20 (95% CI) (66-140) (7.65-15) (66-136) (12-29)(52-124) (16-36) (84-199) (17-33) (95-205) (22-50) (88-127) (17-24) GMR17* 2.11 19* 3.73 16* 4.57 21* 4.59 26* 6.05 19* 3.95 (95% CI) (12-23)(1.6-2.79) (13-27) (2.42-5.76) (11-24) (3.15-6.63) (14-32) (3.39-6.22)(18-36) (4.23-8.64) (16-23) (3.38-4.62) Seroprotection^(a) 100* 12 95*27 100* 38 100* 33 100* 57 99* 33 % (82-100) (2-30) (77-100) (8-55)(85-100) (20-59) (81-100) (17-53) (85-100) (34-78) (95-100) (25-42) (95%CI) HI titre ≧ 160 26** 0 36** 0 32 8 56* 3 57** 14 41* 5 % (95% CI)(9-51) (0-13) (17-59) (0-22) (14-55) (1-25) (31-78) (0.084-17) (34-77)(3-36) (32-51) (2-11) Seroconversion^(b) 100* 12 95* 27 100* 38 100* 33100* 57 99* 33 % (95% CI) (82-100) (2-30) (77-100) (8-55) (85-100)(20-59) (81-100) (17-53) (85-100) (34-78) (95-100) (25-42)

TABLE III Adjuvanted Unadjuvanted A/ A/ H3N2 A/H1N1 B H3N2 A/H1N1 B Pre- 8.08  5.99  5.2 8.53 6.55 5 vaccination GMT Post- 106 * 55 * 11 * 41 266.07 vaccination GMT GMR  13 *  9.11 *  2.12 * 4.78 4 1.21

TABLE IV (Geometric Mean Ratios, Seroprotection and Seroconversion Ratesby Vaccine and Age Group) Number of Subjects (%) and (95% CI) StrainA/H1N1 A/H3N2 B Vaccine Group Sub/MF59 split Sub/MF59 Split Sub/MF59split Population N = 41 N = 40 N = 41 N = 40 N = 41 N = 40 OverallSP^(a) 6 (15%) 2(5%) 36(88%)* 16(40%) 4(10%) 0(0%) (day 1) (6, 29) (1,17) (74-96) (25-57) (3-23) (0-9) SP^(a) 41(100%) 40(100%) 41(100%)40(100%) 41(100%)* 27(68%) (day 22) (91-100) (91-100) (91-100) (91-100)(91-100) (51-81) GMR (day 22/day1) 91 52 17 12 18* 8.14 (59-140) (35-79)(12-24) (8.08-18) (14-24) (5.7-12) Serocon. rate^(b) (day 22) 39(95%)38(95%) 40(98%) 34(85%) 40(98%)* 27(68%) (83-99) (83-99) (87-100)(70-94) (87-100) (51-81) N = 23 N = 20 N = 23 N = 20 N = 23 N = 20 <3years SP^(a) 2(9%) 1(5%) 22(96%)* 10(50%) 1(4%) 0(0%) (day 1) (1-28)(0-25) (78-100) (27-73) (0-22) (0-17) SP^(a) 23(100%) 20(100%) 23(100%)20(100%) 23(100%)* 9(45%) (day 22) (85-100) (83-100) (85-100) (83-100)(85-100) (23-68) GMR (day 22/day1) 122** 43 17*** 7.86 19* 4.14 (77-194)(25-75) (11-24) (5.01-12) (14-27) (2.7-6.35) Serocon. rate^(b) (day 22)23(100%) 19(95%) 23(100%) 17(85%) 22(96%)* 9(45%) (85-100) (75-100)(85-100) (62-97) (78-100) (23-68) N = 18 N = 20 N = 18 N = 20 N = 18 N =20 ≧3 years SP^(a) 4(22%) 1(5%) 14(78%)* 6(30%) 3(17%) 0(0%) (day 1)(6-48) (0-25) (52-94) (12-54) (4-41) (0-17) SP^(a) 18(100%) 20(100%)18(100%) 20(100%) 18(100%) 18(90%) (day 22) (81-100) (83-100) (81-100)(83-100) (81-100) (68-99) GMR (day 22/day1) 63 64 18 19 17 16 (28-141)(34-121) (9.58-34) (9.68-36) (11-26) (11-24) Serocon. rate^(b) (day 22)16(89%) 19(95%) 17(94%) 17(85%) 18(100%) 18(90%) (65-99) (75-100)(73-100) (62-97) (81-100) (68-99) ^(a)Seroprotection: HI titers ≧40 IU;^(b)Seroconversion rate: seroconversion and/or significant increase;Seroconversion - negative pre-vaccination serum (i.e., HI titer <10 IU)and post-vaccination HI titer ≧40 IU and Significant increase - at least4-fold increase in HI titers in subjects who were positivepre-vaccination (i.e., HI titer ≧10 IU). *p < 0.001; **p < 0.01; ***p <0.05 vs. split group

1. A composition in unit dosage form, wherein: the composition comprises(i) an influenza B virus antigen and (ii) an adjuvant; and the unitdosage has a volume of between 0.2 mL and 0.45 mL, wherein the adjuvantcomprises an oil-in-water emulsion in which the majority of oil dropletshave a diameter of less than 1 μm and the oil droplets comprisesqualene.
 2. A composition in unit dosage form, wherein: the compositioncomprises (i) an antigen from at least one influenza B virus strain and(ii) an adjuvant; and the unit dosage contains between 6-9 μg ofhemagglutinin per influenza virus strain, wherein the adjuvant comprisesan oil-in-water emulsion in which the majority of oil droplets have adiameter of less than 1 μm and the oil droplets comprise squalene.
 3. Akit for preparing an immunogenic composition for use in immunizing achild, wherein the kit comprises (i) a first kit component comprising aninfluenza B virus strain antigen and (ii) a second kit componentcomprising an adjuvant, wherein the adjuvant comprises an oil-in-wateremulsion in which the majority of oil droplets have a diameter of lessthan 1 μm and the oil droplets comprise squalene; and wherein theimmunogenic composition has a unit dose of between 0.2 mL and 0.45 mL.4. A kit for preparing an immunogenic composition for use in immunizinga child, wherein the kit comprises a first kit component comprising aninfluenza B virus strain antigen for mixing with a second componentcomprising an adjuvant, wherein the adjuvant comprises an oil-in-wateremulsion in which the majority of oil droplets have a diameter of lessthan 1 μm and the oil droplets comprise squalene; and wherein theimmunogenic composition formed when the first kit component is mixedwith the second component has a unit dose of between 0.2 mL and 0.45 mL.5. A kit for preparing an immunogenic composition for use in immunizinga child, wherein the kit comprises a first kit component comprising anadjuvant for mixing with a second component comprising an influenza Bvirus strain antigen, wherein the adjuvant comprises an oil-in-wateremulsion in which the majority of oil droplets have a diameter of lessthan 1 μm and the oil droplets comprise squalene; and wherein theimmunogenic composition formed when the first kit component is mixedwith the second component has a unit dose of between 0.2 mL and 0.45 mL.6. The composition of claim 1, wherein the composition is for use inimmunizing a child.
 7. The composition of claim 1, wherein thecomposition is for use in immunizing a child.
 8. The kit of any one ofclaims 3-5 or the composition of claim 6 or claim 7, wherein the childis less than 36 months old.
 9. The kit of any one of claims 3-5 or thecomposition of claim 6 or claim 7, wherein the child is at least 6months old.
 10. The kit of any one of claims 3-5 or the composition ofclaim 6 or claim 7, wherein the child is at least 6 months old but lessthan 36 months old.
 11. The kit of any one of claims 3-5 or thecomposition of claim 6 or claim 7, wherein the composition includes asubtype H3N2 influenza A strain antigen.
 12. The kit of any one ofclaims 3-5 or the composition of claim 6 or claim 7, wherein thecomposition includes a subtype H1N1 influenza A strain antigen.